PROJECT SUMMARY Obstructive sleep apnea (OSA) is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep, which leads to intermittent hypoxia and sleep fragmentation. OSA is a common disorder affecting 25-30% of adult population and a major cause of cardiovascular morbidity and mortality. Continuous positive airway pressure relieves OSA, but poor adherence severely limits its use. There is an urgent need for alternative therapies that reverse neuromuscular defects in upper airway function. The upper airway patency is regulated by lingual protrudors, including the biggest upper airway dilator, the genioglossus (GG) muscle, but both protrudors and retractors may act synergistically to stabilize the upper airway during sleep. The development of therapeutic strategies has been hindered by the lack of knowledge about the role of different tongue muscles in the pathogenesis of OSA, since methods to manipulate these muscles selectively have not hitherto been available. We have demonstrated that mouse upper airway physiology is identical to humans and that obese mice develop OSA. We will use our mouse model of OSA and other novel investigative tools, i.e. chemogenetics and dynamic magnetic resonance imaging (MRI), to examine the impact of specific hypoglossal motor neurons innervating lingual protrudor and retractor muscles on pharyngeal patency and obstructive sleep apnea. Our proposal utilizes a novel chemogenetic approach for targeting specific motor neuron populations selectively with designer receptors exclusively activated by designer drugs (DREADDs). Excitatory and inhibitory DREADDs will be used to address the main hypothesis that stimulation of specific populations of hypoglossal motor neurons is both necessary and sufficient to stabilize pharyngeal patency and treat OSA. Specific Aim 1 and 2 will examine the role of different populations of hypoglossal motor neurons in maintaining upper airway patency and the development of OSA. We hypothesize that (A) chemogenetic stimulation of GG fibers in isolation OR in combination with retractor muscles will increase patency of the upper airway and treat OSA, whereas (B) chemogenetic inhibition will decrease airway patency and induce OSA. Cre-dependent DREADDs will be deployed in the hypoglossal nucleus and selectively expressed in motor neurons innervating tongue protrudors alone OR protrudors and retractors using Cre-recombinase administered with a retrograde neuronal vector. In Specific Aim 1, dynamic MRI will be performed before and after motor neuron manipulation by a DREADD specific ligand, clozapine-N- oxide (CNO), in lean and obese male and female mice. In Specific Aim 2, sleep studies will be performed with or without DREADD activation by CNO to identify the effect of chemogenetic manipulation of different motor neuron pools on sleep apnea during NREM and REM sleep. The proposal will focus future therapeutic strategies on specific hypoglossal motor neurons for electrical and pharmacological stimulation.